J Mammary Biol Neoplasia (2010) 15:301–318 DOI 10.1007/s10911-010-9189-6

Extracellular Composition Reveals Complex and Dynamic Stromal-Epithelial Interactions in the

Ori Maller & Holly Martinson & Pepper Schedin

Received: 30 July 2010 /Accepted: 16 August 2010 /Published online: 2 September 2010 # Springer Science+Business Media, LLC 2010

Abstract The mammary gland is an excellent model the context of the morphologically-distinct stromal sub- system to study the interplay between and epithelial compartments: the , the intra- and interlobular cells because of the gland’s unique postnatal development stroma, and the fibrous connective . Future studies and its distinct functional states. This review focuses on the aimed at in-depth qualitative and quantitative characteriza- contribution of the (ECM) to stromal- tion of mammary ECM within these various subcompart- epithelial interactions in the mammary gland. We describe ments is required to better elucidate the function of ECM in how ECM physical properties, composition, and normal as well as in pathological tissue. proteolytic state impact mammary gland architecture as well as provide instructive cues that influence the function Keywords Extracellular matrix . Stromal-epithelial of mammary epithelial cells during pubertal gland devel- interactions . Basal lamina . . . opment and throughout adulthood. Further, based on recent proteomic analyses of mammary ECM, we describe known mammary ECM and their potential functions, as Abbreviations well as describe several ECM proteins not previously collagen IV type IV collagen recognized in this . ECM proteins are discussed in DDR1 discoidin domain 1 EM electron Financial Support Supported by Department of Defense Idea EGFR epidermal receptor Award#BC095850 to PS. ECM extracellular matrix : : O. Maller H. Martinson P. Schedin (*) ED extra domain Department of Medicine, Division of Medical Oncology, FACIT associated with University of Colorado Denver, interrupted triple helices MS8117, RC-1S, 8401K, 12801 E 17th Ave, Aurora, CO 80045, USA FGF growth factor e-mail: [email protected] FN fibronectin : : GAG O. Maller H. Martinson P. Schedin H&E hematoxylin and eosin stain Program in Biology, University of Colorado Denver, MS8104, RC-1S, 5117, 12801 E 17th Ave, HGF growth factor Aurora, CO 80045, USA LN laminin LAP latency-associated P. Schedin LRR leucine-rich repeats University of Colorado Cancer Center, Bldg 500, Suite 6004C, 13001 E 17th Place, LOX lysyl Aurora, CO 80045, USA MMP MAGP -associated P. Schedin MCP-1 monocyte chemoattractant protein-1 AMC Cancer Research Center, Bldg 500, Suite 6004C, 13001 E 17th Place, SPARC secreted protein acidic and rich in Aurora, CO 80045, USA SLRP small leucine-rich 302 J Mammary Gland Biol Neoplasia (2010) 15:301–318

SD Sprague Dawley heterodimerize to generate 24 canonical receptors TN [11, 12]. A comprehensive description of ECM-integrin tTG2 2 interactions is beyond the scope of this review; the reader is TGF-β transforming growth factor β referred to reviews by Desgrosellier et al. and Larsen et al. TLR toll like receptor [13, 14]. ECMsynthesisandassemblyinthemammarygland are the product of many types including epithelial cells, myoepithelial cells, , , endo- Introduction thelial cells, and immune cells [15–17]. Bissell and colleagues describe the relationship between ECM and The mammary gland is an excellent model system to study mammary epithelial cells as dynamic and reciprocal, the function of stromal-epithelial interactions because of the leading to two-way communication, where ECM instructs gland’s unique development across its several distinct and supports cells, while cells build, shape, and re-shape functional states, depending on reproductive state. Unlike the ECM [18]. Mammary ECM can be separated into three most organs, which develop to morphological maturity broad structural categories: the highly-specialized basal during embryogenesis, the majority of mammary ductal lamina that directly abuts the basal side of mammary occurs with the onset of ovarian function. epithelial cells (Fig. 1), the intra- and interlobular stroma Further, with each estrous (rodent) or menstrual (human) immediately adjacent to alveoli and lobules, respectively cycle, the alveoli undergo cyclic expansion and maturation, (Figs. 1 and 2), and the fibrous that is followed by a modest regression phase as ovarian hormone devoid of (Fig. 2). In this review we explore levels rise and fall, respectively [1–4]. The mammary gland the composition and function of each of these ECM is also unique in that terminal differentiation does not occur categories using data obtained largely from rodent models. unless pregnancy and lactation ensue [5]. These unique Although rodent models recapitulate many aspects of attributes highlight the dynamic, non-homeostatic nature of human mammary gland biology, there are interspecies the normal mammary gland. variations in mammary stroma composition, organization, Since mammary gland development is largely postnatal, density, and function. Thus, while significant insight into organogenesis and differentiation are extended in time, mammary ECM is obtained through the study of rodents, occurring over a period of weeks in the rodent, rather than future studies validating results in human breast tissue are hours and days as observed in other organs. Further, in essential. rodents, the development of mammary occurs along the entire flank of the with 5 pairs of glands in the mouse and 6 pairs in the rat. As a result, there is sufficient Rodent Mammary Gland as a Model System to Study mammary tissue in these species to permit physical, Human Breast functional, and biochemical characterizations. These fea- tures make the rodent mammary gland a highly suitable The mouse and rat models recapitulate critical aspects of model to evaluate the interplay between stroma and human mammary gland development and function. The epithelium throughout gland development and during rodent mammary gland and human breast have similar distinct functional states such as pregnancy or lactation. rudimentary mammary , which consist of a The focus of this review is on the extracellular matrix layer of luminal epithelial cells, a basal epithelial cell layer (ECM) component of the mammary stroma. ECM can be that putatively contains mammary stem cells, and the outer described as an interconnected meshwork of secreted contractile myoepithelial cells adjacent to the basal lamina proteins that interacts with cells to form a functional unit ECM [19]. The parenchyma structure consists of a single [6]. Cell-ECM interactions have been implicated in cell elongated ductal tree (rodent) or multiple ductal networks adhesion, survival, , polarity, proliferation, and (human) that develop by bifurcation of specialized end bud differentiation. ECM confers these cellular functions through structures present during puberty [20]. While the overall structural properties such as physical support and boundary epithelial structure is similar between humans, rats, and constraints, classical pathways, and by mice, the relative abundance of connective tissue varies providing mechanosensory cues [7]. Epithelial cells receive considerably. Stroma surrounding the lobules and ducts instructive cues from the ECM via integrin receptors and (intra- and interlobular stroma) in mice is sparse and there the non-integrin receptors such as discoidin domain is little acellular fibrous connective tissue between ducts. receptor 1 (DDR1) [8], [9], and Instead, the mammary glands of mice are characterized by [10]. are transmembrane receptors composed of α an abundance of white that is directly and β subunits, with 18 α and 8 β subunits that adjacent to the sparse interlobular stroma (Fig. 2c). In J Mammary Gland Biol Neoplasia (2010) 15:301–318 303

host exposures are unlikely to completely explain variation in stromal density given that very distinct microenviron- ments can exist within individual adjacent lobules (Fig. 3). Secretory epithelial cell Pleiotropic Roles for ECM in the Mammary Gland

The ECM provides physical support that is essential for BL overall tissue architecture. Changes in ECM properties are communicated to epithelial cells via transmembrane recep- ULLC tors that provide a conduit between the ECM, the Fibroblast and its transcriptional machinery [25]. Thus, ECM interactions ultimately determine epithelial cell phenotype. A role for ECM in the mammary Lumen gland has been increasingly explored including the role of stiffness requirements for establishing apical/basal and lateral cell junctions [26], as well as for [27, 28]. Importantly, Discher and colleagues have determined that matrix stiffness and mechanotrans- duction are seminal factors in mesenchymal lineage specification [29]. By modulating matrix stiffness in vitro, this group demonstrated that mesenchymal stem Figure 1 Electron micrograph image of mammary epithelial cells cells acquired a neuronal profile on soft matrix, while from a lactating mouse. The solid blue arrow points to the basal acquiring -like properties on rigid matrix [29]. In lamina (BL) adjacent to a putative mammary progenitor cell known as β “ undifferentiated large light cell (ULLC), and a secretory epithelial cell. the mammary gland, 1 integrin is a cellular mechano- The dashed red arrow points to intralobular fibrillar collagen adjacent sensor” that is imperative for maintaining basal progenitor to a fibroblast. Scale bar represents 5 microns cells, as demonstrated by inability of mammary epithelium lacking β1 integrin to repopulate the gland upon serial transplantation [30]. These data suggest that physical humans the ratio of fibrous connective tissue to adipose properties and site-specific composition of adjacent basal tissue is opposite, with an abundance of stroma surrounding lamina ECM may contribute to the “stemness” of mammary the alveoli and ducts, a predominance of fibrous connective progenitor cells through β1 integrin. tissue between ducts, and reduced adipose content (Fig. 2a). In order to understand how ECM stiffness influences the Organization of the stroma in outbred Sprague Dawley function and fate of mammary epithelial cells, it is vital to (SD) female rats is intermediate between mice and humans understand its composition as well as its architecture, which and thus is histologically more similar to humans than is the is largely defined by protein-protein interactions. While mouse (Fig. 2b)[21]. While the mechanisms driving the secreted ECM proteins can interact through numerous, different ratios of fibrous connective to adipose tissues weak non-covalent bonds, crosslinking these proteins via across species are currently unknown, we suggest a covalent chemical bonds is essential for ECM scaffold relationship to ovarian hormone exposure, particularly organization, tensional characteristics, and stabilization. progesterone, may exist. Compared to mice, the SD rat (LOX), a copper-dependent , cata- has a robust luteal phase during the estrus cycle, resulting in lyzes intra- and intermolecular crosslinking of fibrillar cyclical progesterone exposure that is more similar to that collagens and elastic fibers through oxidative deamination for women [1]. Given that pituitary and ovarian hormone of residues [28, 31, 32]. LOX activity has been exposure drives most aspects of gland development and shown to promote tissue stiffness and as well as adult function, instructive cues from hormones are expected modulate elastic properties of elastic fibers, establishing to also directly or indirectly influence composition and LOX as a key regulator of tissue tension [28, 31–33]. organization of the adjacent stroma [22–24]. Hence, Tissue transglutaminase 2 (tTG2) has also been implicated studying the hormonal regulation of normal mammary in crosslinking ECM proteins in the mammary stroma. The gland biology may afford novel insights into how stromal crosslinking activity of tTG2 depends on calcium and organization is regulated under physiological conditions, proceeds by catalyzing covalent bonds between glutamine which can be utilized to decipher this relationship in terms and ε-amino groups of lysine residues [34]. Future studies of tissue plasticity and breast disease. However, systemic/ are needed to examine how LOX and tTG2 are regulated in 304 J Mammary Gland Biol Neoplasia (2010) 15:301–318

Human Breast Mammary Gland of Mammary Gland of SD Rat C3H Mouse abc

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Figure 2 Histological sections from human breast and rodent stroma. Open arrow heads point to adipocytes, which are abundant in mammary gland from nulliparous females. a–c H&E-stained nullip- rat and mouse glands. d–f Serial sections stained with Masson’s arous human breast tissue (a), nulliparous SD rat mammary tissue (b), trichrome showing collagen as blue fibers in human nulliparous breast and nulliparous C3H mouse mammary tissue (c). In a, solid arrow tissue (d), nulliparous SD rat mammary tissue (e), and nulliparous head points to fibrous connective tissue (pink) that is dominant in C3H mouse mammary tissue (f). Scale bar represents 100 microns human breast tissue. Arrow in the inset box points to intralobular the mammary gland and to determine their unique GAGs [20, 36]. These observations are interesting because contributions to ECM function. the terminal end bud tips are actively elongating structures while the flank of the end bud is not [37]. These observations raise questions on how GAG composition contributes to ECM as a Reservoir and a Source for Growth Factors ECM thickness, rigidity, and the ability of ECM to serve as a and Cytokines reservoir for growth factors and cytokines, as well as the ability of epithelial cells to penetrate into the surrounding Growth factors and cytokines can bind to ECM proteins, stroma and proliferate. identifying another avenue by which ECM can regulate Release and activation of growth factors and cytokines mammary morphogenesis and function. For example, trans- from ECM can occur by altering matrix stiffness and/or gluaminase crosslinks the large latent complex of trans- inducing ECM [38, 39]. For example, TGF-β forming growth factor β (TGF-β), known as latent TGF-β activity can depend on fibrillar ECM proteolysis or altered binding protein, to ECM proteins [35]. Growth factors and matrix stiffness. TGF-β is secreted as a large latent cytokines bind to ECM through (GAG), complex (LLC) by multiple cell types and associates with which are anionic linear consisting of fibrillar ECM proteins [40]. TGF-β is activated by repeating hexuronic acid and hexosamine. GAG can be proteolytic cleavage of the latency-associated peptide thought of as “glue” that allows growth factors to “stick” to (LAP) or by conformational change in LAP through basal lamina and fibrillar ECM proteins. Interestingly, binding to the αvβ6 integrin receptor [41, 42]. However, different classes of GAGs are associated with distinct aspects Hinz and colleagues recently published evidence suggesting of mammary ductal elongation [20]. Silberstein et al. and that mechanical tension is also involved in the release of Williams et al. used electron microscopy (EM) to show that TGF-β1 from the ECM [43]. They suggested that high the basal lamina surrounding the tips of end buds in mice is contractile activity of on a stiff matrix may thin and rich in , while the basal lamina along lead to a conformational change in LAP, resulting in TGF- the flank of end buds is thicker and associated with sulfated β1 release, while on soft matrix the latent complex stays J Mammary Gland Biol Neoplasia (2010) 15:301–318 305

Human Breast β(s) is significantly downregulated [48], which may prevent TGF-β(s) from negatively regulating expression of milk proteins such as β-casein [48, 50, 51]. Upon the onset of involution when the gland remodels toward its pre- pregnant state, there is an upregulation of TGF-β tran- scripts, particularly for TGF-β3[52]. TGF-β signaling may further contribute to the remodeling of the involuting gland H&E by inducing ECM production, upregulating MMP expres- sion, and by recruiting immune cells [53–55]. Taken together, TGF-β(s) expression and activation are tightly regulated in mammary gland development where they mediate stromal-epithelial interactions, in part by orches- trating ECM deposition and remodeling. Instructive cues are also provided to cells from the ECM Figure 3 H&E-stained nulliparous human breast tissue. This histo- by the controlled release of -cleaved ECM frag- logical section demonstrates how adjacent lobules can exist in very ments, termed ‘matricryptins’ [56, 57]. These fragments distinct microenvironments. The arrow points to fibrous intralobular exhibit altered biological functions compared to intact ECM stroma and the arrow head points to -rich stroma. Scale bar proteins. For example, fibronectin (FN) fragments initiate represents 100 microns apoptosis in epithelial cells [58], while intact FN promotes and proliferation [59, 60]. A subclass of intact due to lack of mechanical stretch [43]. Additional matricryptins, “matrikines,” are short from ECM support for mechanical tension regulating ECM function is proteins that function in a similar manner as cytokines [56, presented in studies demonstrating how fibroblast traction 57]. Instructive cues from ECM fragments will be further forces lead to large-scale directional patterning of fibrillar discussed below in the context of immune cells and ECM ECM proteins such as collagen І [44, 45]. Given that ECM remodeling. tensional requirements and organization are expected to change throughout mammary gland development, it is likely that the ability of ECM to serve as a reservoir for ECM Composition in the Mammary Gland growth factors and cytokines would be altered accordingly. During expansion of the ductal network, Daniel and In order to understand the myriad of functions mediated by the colleagues postulated that TGF-β1 regulates branching ECM, one must have an in-depth understanding of the ECM morphogenesis by inhibiting ductal growth through direct proteins that contribute to each of the distinct ECM suppression of epithelial cell proliferation and by stimulat- subcompartments. Our current understanding of ECM com- ing production of collagen I, GAG, and position is rudimentary and largely derives from studies of within the periductal stroma at puberty [46]. Evidence for rodent mammary gland tissue (mouse and rat). Until recently, this proposal was obtained by administering TGF-β1 the standard method for identifying ECM proteins was by directly into the mammary gland of young female mice, biochemical isolation and Edman sequencing and/or by which resulted in premature inhibition of ductal elongation antibody-based detection of candidate proteins. Mass concurrent with aberrant and increased deposition of ECM spectrometry-based proteomic analysis provides an unbiased, proteins [46, 47]. Moreover, as discussed above, these high-throughput comprehensive overview of ECM composi- researchers observed high expression of TGF-β3 in the tion. Unfortunately, the traditional digestion approaches flank region of end buds, suggesting it may positively required to prepare proteins for proteomic analysis have regulate ECM production at this site [48]. Given the growth proven ineffective for ECM proteins due to their resistance to suppressive role of TGF-β during pubertal gland develop- proteolysis. In the past few years, Liang and others have used ment, it was surprising that expression of TGF-β2 and media conditioned by mammary epithelial cells in vitro to TGF-β3 transcripts was elevated during pregnancy [48]. As analyze their secretomes and have identified peptides origi- mentioned by Robinson et al. [48], this observation is not nating from ECM proteins such as FN, laminin (LN), and type consistent with the growth inhibitory function that TGF- IV collagen (collagen IV) [61]. An in-solution digestion β(s) has on mammary epithelial cells during ductal approach for trypsin-resistant ECM proteins was recently elongation. A later study suggested that TGF-β1 only developed by Hansen et al., permitting the identification of impedes epithelial in ducts, but not in alveoli, known and previously unknown ECM proteins within the rat although the mechanism of this differential effect is mammary gland [62]. In the next section of this review, we unknown [49]. During lactation, the expression of TGF- describe many of the well-known ECM proteins in the 306 J Mammary Gland Biol Neoplasia (2010) 15:301–318 mammary gland and highlight several ECM proteins not LN111 is one of the major constituents of the basal previously recognized. We discuss these ECM proteins in the lamina and was the first LN characterized. LN111 was context of the three distinct subcompartments depicted in isolated from a poorly-differentiated murine sarcoma Figs. 1 and 2. (Engelbreth-Holm-Swarm (EHS) sarcoma), and is a princi- pal component of , a commercially-available Basal Lamina substitute [68, 69]. LN111 is neces- sary for cell differentiation as documented in murine The terms basal lamina and basement membrane have been embryogenesis, where loss of any LN111 chain (α1, β1, used interchangeably to refer to the highly specialized ECM or γ1) causes embryonic lethality due to improper basal directly underlying the epithelium. While basement mem- lamina assembly and function [70, 71]. An insightful brane refers to the ECM adjacent to the epithelium that is example was obtained after deletion of specific domains visible by light microscopy, the basal lamina is a sub-light in the C-terminus of the LN α1 chain [9]. The C-terminus microscope structure visualized only by EM. The basal of the LN α1 chain consists of 5 globular domains with lamina lies immediately beneath the epithelium and is a known cell binding motifs [72]. Deletion of the α1 globular highly organized 20–100 nm thick structure (Fig. 1). Basal 4–5 domains, which are binding sites for the ECM receptor lamina in the mammary gland is thicker along the flanks of dystroglycan, impeded pluripotent epiblast differentiation the terminal end bud compared to the growing bud tip, [9]. Interactions between α1 globular 4–5 domains and which correlates with compositional and possibly function- dystroglycan are crucial for gastrulation and for the al differences, as mentioned above [36]. The basal lamina formation of the primary germ layers, likely because LN consists of three layers: the middle electron dense layer is needed for epithelial cell polarization [9]. Using a 3D termed the , and the inner and outer less-dense culture model, Bissell and colleagues showed that LN111- electron layers referred to as the externa and rich reconstituted basement membrane is pivotal for interna, respectively [63]. The lamina lucida layers contain apicobasal polarization of mammary epithelial cells and high levels of (hence they appear as clear or for lumen formation of acini [73–75]. Cultured luminal ‘lucid’ areas) that decorate a meshwork of small filaments mammary epithelial cells did not form acini in collagen I that attach the lamina densa to the epithelial cell surface, gels unless the cells were mixed with myoepithelial cells and to the collagen within the stroma on the basal [76]. Petersen and colleagues identified myoepithelial cells side. This creates a highly organized ECM layer between as the primary source of LN111 via in vitro assays and the epithelial cell and its underlying stroma. The major through staining of human breast tissue [76]. The in vitro constituents of basal lamina are LN, collagen IV, , studies have demonstrated that LN111 instructs acinar and . formation, in part by facilitating deposition of a mammary-specific basal lamina, given that newly- LNs are large heterotrimeric synthesized LN332 and collagen IV are incorporated into (~900 kDa) found in at least 15 different combinations that a basal lamina-like structure in a 3D culture model in the are derived from five α, three β, and three γ subunits, presence of LN111 [77, 78]. coded from distinct [64]. Each LN chain includes LN332 is expressed by mammary epithelial cells and can globular, , and rodlike regions [65]. The three induce adhesive contacts in epithelial cells via α6β4or distinct chains are intertwined at the coiled coil regions by α3β1 integrins [79, 80]. Moreover, MMP2-cleaved LN332 bonds to form a cross-shape structure [65]. LN fragments (from the γ2 chain) have been shown to bind the deposition appears to be restricted to the region of the basal epidermal growth factor receptor (EGFR) [81]. LN332 lamina, is in direct physical contact with epithelial cells, fragment-EGFR interaction was associated with pro- and thus is likely critical mediators of epithelial cell-ECM migratory effects on mammary epithelial cells as well as interactions in vivo. Different LNs can be associated with downstream activation of EGFR signaling, although the specific tissue compartments or organs. For example, LN8 results from the cell growth assay were inconclusive [81]. (α4, β1, γ1) is expressed by endothelial cells and Further, by culturing freshly-isolated murine mammary contributes to the vascular basal lamina [15, 66], while epithelial that included luminal epithelial and LN10 (α5, β1, γ1) is associated with villus cells of the myoepithelial cells in a 3D culture model, Werb and small intestine [67]. These and other examples suggest that colleagues demonstrated that the front edges of actively- particular LNs may be coupled with tissue-specific differ- elongating ducts were devoid of intact LN332 [80]. entiation or function. In this review, we will focus on Moreover, LN332 fragments are present in the mammary LN111 (α1, β1, γ1; known as LN1) and LN332 (α3, β3, gland during mid-pregnancy and involution, suggesting that γ2; previously known as LN5) due to their established LN332 contributes to the that occurs presence in the mammary gland. during these stages [81, 82]. Using a pan anti-LN antibody, J Mammary Gland Biol Neoplasia (2010) 15:301–318 307

LN fragments were apparent in mammary tissue during study, Kidwell and colleagues demonstrated that mammary early involution in the rat [23]. Nevertheless, the functional epithelial cells preferentially attached to collagen IV verses consequences of LN turnover during involution are still collagen I in vitro [89]. Hence, collagen IV is essential for unknown. While LNs experience turnover during early supporting basal lamina structure and provides an anchor involution of the gland [23], LN transcript and total protein for mammary epithelial cells, indicating its specific role in levels change only modestly with pregnancy, lactation and maintaining cell viability. On a different note, cryptic involution, suggesting a fundamental requirement for LNs domains of type IV collagen fragments were demonstrated in mammary epithelial maintenance, rather than for stage- to function as anti-angiogenic factors. is a specific functions. fragment from the C-terminus of collagen IV α3 chain and inhibits by blocking αVβ3integrin Collagen IV Collagen IV is also a major component of the signaling [90, 91]. Further, during early to mid-involution, basal lamina and is thought to be its primary scaffold alveolar loss is associated with both basal lamina remodel- protein [16, 83]. Unlike fibrillar collagen, collagen IV is a ing and a decrease in vascular support needed for lactation network-forming collagen. It is a heterotrimer and can be [90, 92]. Thus, there may be a link between basal lamina comprised from six possible genetically distinct α chains. turnover and vascular regression that occurs during early to Collagen IV α chains trimerize to form three distinct mid-involution. ‘protomers’ that can be distributed in a tissue-specific manner [84]. Two collagen IV protomers bind through the Nidogens (also known as entactins) are sulfated C-terminal non-collagenous 1 (NC1) domain to form a glycoproteins (150 kDa) synthesized by fibroblasts as in hexamer [16]. Hexamers interact with one another through various tissues such as , , central nervous system, and N-terminal 7 S domains to generate a tetramer [16]. limb [93–95]. There are two genes in the nidogen family that Yurchenco and colleagues demonstrated that the lateral encode nidogen 1 and nidogen 2. Nidogens are incorporated interactions among tetramers created a polygonal network into the basal lamina upon secretion [93, 96]. Germ layer of collagen IV and a 3D mesh [16, 85]. The α1.α1.α2 cooperation in the assembly of basal lamina is highlighted by protomer was suggested to form the initial sheet-like basal the fact that mesodermal-derived fibroblasts make nidogen lamina during embryogenesis [84]. Disruption of the COL4 and ectoderm/endoderm-derived epithelial cells produce α1orα2 in mice is embryonic lethal [84]. LNs. Nidogen is found in a 1:1 stoichiometric ratio with Surprisingly, in collagen IV-deficient mice, LN111 and LN and can be viewed as a stabilizing component of the nidogen 1 were deposited and organized into a basal basal lamina by serving as a bridge between LN111 and lamina-like structure, during early embryogenesis (prior to collagen IV [97]. For example, the globular domain G3 of E10.5). The basal lamina in these mice had a discontinuous nidogen 1 binds to the C-terminal short arm of a LN γ1 and altered structure, suggesting that collagen IV is not chain and the G2 domain of nidogen binds collagen IV [98, required for LN111 and nidogen assembly but is required to 99]. Nevertheless, deletion of nidogen 1 in mice demonstrat- stabilize the nascent basal lamina [84]. Furthermore, other ed that it was not essential for basal lamina assembly [100], collagen IV α chains did not compensate for loss of the as nidogen 2 appeared to compensate [101]. Furthermore, α1.α1.α2protomer[84]. Later in development, the mice lacking both nidogen 1 and nidogen 2 do not die during α1.α1.α2 protomer is replaced with different collagen IV embryogenesis, indicating that nidogens are not necessary protomers such as α3.α4.α5 in the glomerular basal lamina for basal lamina assembly in most organs. However, [86]. Evidence for the importance of protomer tissue from newborn mice deficient in both nidogens had small specificity has been obtained from patients with Alports alveolar spaces, thicker septa, and a prenatal decrease in syndrome who have a in the COLα5 chain. This expression of basal lamina constituents such as LN γ1chain, mutation leads to absence of the α3.α4.α5 protomer (IV) which recovered to control levels by the perinatal period based network in the glomerular basal lamina, impeding the [102]. In addition, the ability of type II pneumocytes to required switch from the fetal α1.α1.α2 protomer to the produce pulmonary surfactant may be impaired given that α3.α4.α5 protomer (IV) [86]. Alports syndrome leads to the expression of surfactant protein B was reduced in double progressive renal failure among other conditions [87]. knockout newborn mice [102]. Pulmonary surfactant is Although it is unclear which α chains comprise collagen important in lowering the alveolar surface tension as well IV in the mammary gland, collagen IV deposition has been as maintaining alveolar structure. Taken together, these data found necessary for proper formation of the mammary suggest that nidogen 1 and nidogen 2 are needed for proper lamina densa. Disruption of collagen deposition in the basal function of alveolar epithelial cells in the lung. Their absence lamina in vivo resulted in collapsed alveolar structures in in the lung may be the reason mice lacking both nidogens proliferative mammary epithelium, resulting in a phenotype die shortly after birth [102]. Nidogens also support tissue- similar to that seen during early involution [88]. In another specific differentiation as nidogen 1 promotes the ability of 308 J Mammary Gland Biol Neoplasia (2010) 15:301–318

LN111 to induce β-casein expression by mammary epithelial ECM dictating intra- and interlobular stroma organization cells in vitro [103]. Collectively, these and other data suggest and functions. a functional relevance for nidogens in a tissue-specific manner, but surprisingly, also indicate that nidogens are not Fibrillar Collagens Collagen I, III and V are the major essential for basal lamina formation during embryogenesis. fiber-forming collagens, where their individual proteins organize into bundles of various thicknesses and lengths to Perlecan Perlecan is a proteoglycan that consists of a form the pink or blue fibrous material observed in H&E 470 kDa core protein with covalently-linked heparan (Fig. 2a–c) and trichrome-stained (Fig. 2d–f) mammary sulfate, a specific-class of GAG, which increases the sections, respectively. Collagen І has been demonstrated to molecular weight (MW) of perlecan to over 800 kDa be synthesized by fibroblasts, , osteoclasts, [104]. Analysis of perlecan distribution during development and during normal development and by revealed its expression in the basal lamina of liver, lung, the activated fibroblasts during wound . Thus, mesen- vascular system, and other tissues as well as in interstitial chymal cells are thought to be the primary source of stroma such as the interterritorial ECM of articular collagen І. The collagen I precursor, procollagen (500– [105]. Further, mammary epithelial cells have been shown 600 kDa) consists of two identical α1 (I)-chains and one α2 to secrete proteoglycan, suggesting epithe- (I)-chain, each derived from a unique gene. Each α chain is lial cells as a possible source of perlecan [17]. Disruption of made up of approximately 300 Gly-X-Y repeats where X is the gene encoding perlecan (Hspg2) caused embryonic frequently , and Y is [32, 111]. The lethality in a large portion of mice. Lethality occurs at Gly-X-Y repeats are essential for ‘self-assembly’ of E10.5 due to defective cephalic development and effusion collagen chains into a triple helix procollagen structure, of to the pericardium, which is thought to result from where all the face inward while the larger amino basal lamina deterioration [106, 107]. The remaining acids such as proline cover the outer positions [111, 112]. perlecan-null mice died perinatally with severe skeletal After assembly into the triple helix, peptidases remove non- anomalies, presumably due to the role of perlecan in helical registration peptides from the secreted procollagen matrix (as described above) [106, 107]. Moreover, Fässler [111] and subsequently it is transformed into insoluble and colleagues observed discontinuous areas in the basal 300 nm tropocollagen [111]. At the end of each triple helix lamina in vivo that separated the neuroepithelium from the tropocollagen molecule there are short non-helical telopep- underlying [106]. All together, the phenotypes tides. These allow further stabilization and aggregation by of the null mice suggest that perlecan is essential for basal covalent intra- and intermolecular crosslinking via LOX lamina assembly in several organs and for bone matrix resulting in the basic fibrillar structure of collagen observed function. Additionally, perlecan’s soluble C- terminal in histological sections [32, 111]. Based on its ubiquitous fragment, endorepellin, disrupted morphogenesis and abundant presence, fibrillar collagen І is considered the in vitro by inhibiting endothelial , and backbone of stroma, including that in the mammary gland. angiogenesis [108]. Moreover, perlecan mediates the effects As such, the physical and biochemical properties of of several (FGF) family members. collagen І likely contribute significantly to the functional Perlecan was suggested to interact with FGF2 through its attributes of the mammary stroma. core protein and GAG group [109]. These interactions can Work from the Taylor-Papadimitriou and Sonnenschein enhance the potency of FGF2 signaling through FGFR1 labs demonstrated that collagen I supported mammary and FGFR3 in vitro and consequently have a pro- ductal formation in 3D culture [113, 114]. Further, when angiogenic effect [109, 110]. As with most ECM proteins, the collagen-specific α2 integrin subunit was knocked out perlecan provides instructive cues dependent on context in vivo, branching complexity in mammary glands was and structural integrity. reduced [115]. Nonetheless, the epithelial layer in vivo is separated from the intralobular stromal ECM by the basal lamina. Therefore, how do these ECM subcompartments Intra- and Interlobular Stroma and stromal cells such as fibroblasts cooperate to influence mammary epithelial morphogenesis? Krause et al., embed- Immediately adjacent to the basal lamina are 10–100 μm ded mammary epithelial cells with and without fibroblasts thick bands of organized, collagen-rich stroma, known as in collagen I gels and in Matrigel-collagen I gels [114]. intralobular stroma, that surround individual alveoli Epithelial cells alone could only form ductal structures in (Fig. 2a, arrow in the insert box). Clusters of alveoli that collagen I gels in comparison to Matrigel-collagen I gels, compose a single lobule are further surrounded by bands of however when co-cultured with fibroblasts, the epithelial stroma referred to as interlobular stroma. Fibrillar collagens cells formed ductal structures in both gel conditions [114]. are the dominant structural constituents, with several other Moreover, fibroblast-secreted factors such as hepatocyte J Mammary Gland Biol Neoplasia (2010) 15:301–318 309 growth factor (HGF) can enhance the ability of mammary structures characteristic of fibrillar collagen, these collagens epithelial cells to form duct-like structures in collagen І gels have features distinct from collagen I. For example, localized [113]. Collectively, these studies demonstrate that inter- helix instability in collagen III can be caused by the higher plays between fibrillar collagens, integrins, and growth content of in α1 (III) chains [32]. This structural factors are required for mammary duct development. feature may cause an increase in the turnover rate of collagen Fibrillar collagen-epithelial cell interactions are also III and subsequently the collagen III matricryptins may serve critical for supporting the mammary epithelium during as potent chemoattractants for innate immune cells. Further- pregnancy and lactation. Collagen-associated receptor, more, collagen III is a homotrimer consisting of a single α DDR1 may control the tremendous increase in epithelial chain, while collagen V is a heterotrimer composed of three proliferation that occurs during pregnancy [8]. DDR1-null different α chains [111]. These variations in primary mice demonstartaed hyperproliferation of the mammary structure likely cause additional changes in biochemical epithelium during pregnancy and were unable to lactate, properties, as fibrillar collagen I, III, and V interact with one despite the ability of luminal epithelial cells to express another to assemble into supramolecular collagen bundles normal transcriptional levels of milk proteins [8]. Hence, [111]. The assembly of these bundles is further supported by interrupting the collagen-DDR1 interaction causes misguid- short triple-helical fibril associated collagens with interrupted ed instructive cues that are imperative for proper alveolar triple helices (FACIT) collagens such as collagen ІX, which expansion and secretory function [8]. can function as crosslinking bridges [32, 111]. Interactions With cessation of milk secretion and weaning, the between fibrillar collagens and FACIT collagens may be mammary epithelium involutes and returns toward its important for mammary gland remodeling by dictating pre-pregnant, non-secretory state. The stroma in the collagen organization and turnover. involuting gland shares attributes with fibrotic, desmo- In summary, the function and expression levels of plastic stroma seen during . During fibrillar collagens are altered during the various stages of involution, fibrillar collagens are upregulated in the mammary gland development. These studies suggest mammaryglandandaninfluxofinnateimmunecells unique roles during duct formation, alveolar expansion such as are observed [116–118]. Based on during pregnancy, as well as to the demoplastic-like gene array data, the onset of involution is characterized by microenvironment during involution, highlighting the dy- elevated expression of several fibrillar collagens including namic role of fibrillar collagens in the mammary gland. collagen І,III,andVaswellasanincreaseinthe However, even for ECM proteins as ubiquitous as fibrillar expression of genes involved in ECM turnover [23, 52]. collagens, our basic understanding of both function and We and others have demonstrated that non-fibrillar mechanism of action remains largely unknown. denatured collagen can be a positive chemoattractant for macrophages in vitro [118]. Although it is unclear whether Fibronectin FN is a large dimeric glycoprotein (~500 kDa) macrophages are necessary for collagen accumulation or that mediates cell adhesion, migration, proliferation, and remodeling of the mammary ECM during involution, branching morphogenesis. FN is encoded by a highly- monocyte chemoattractant protein 1 (MCP-1) increases conserved single gene and its deletion causes embryonic in early to mid-involution [118]. The presence of MCP-1 lethality during gastrulation, emphasizing its essential was shown to be important for collagen fiber formation in function [120]. FN consists of three distinct repeating a chemical-induced skin fibrosis model, as MCP-1 null modules known as type І, II, and III domains [121]. mice were resistant to chemical-induced skin fibrosis, Although FN protein is encoded by a single gene, there are including fibrillar collagen deposition [119]. Further, the several variant FN forms that arise due to alternative loss of MCP-1 was associated with a decrease in macro- splicing [121, 122]. Specific examples are the spliced sites phages and other innate immune cells, in addition to an in the type III domain, termed extra domain (ED) EDA, increase in the small leucine-rich proteoglycan (SLRP) EDB, and IIICS. Extracellular stimuli such as HGF have . The relationship between SLRP and collagen been shown to regulate splicing in vitro [122]. EDA and fibrillogenesis will be discussed later in this review. EDB have been implicated in wound healing, as their Collectively, the data suggest that fibrillar collagen- expression is undetectable in normal liver but is rapidly interactions may, in part, drive the tissue upregulated during injury [123]. In addition to its essential remodeling programs within the involuting mammary role in early embryogenesis, FN is an essential regulatory gland. protein for salivary gland branching morphogenesis [124]. Another open question is whether unique functions are Yamada and colleagues blocked cleft formation and imparted by distinct fibrillar collagens with respect to collagen branching in the salivary gland ex vivo by downregulating bundle formation, ECM turnover, and cell-collagen interac- FN expression in salivary epithelial cells via siRNA or by tions. Although collagens III and V form oriented super- inhibiting FN-epithelial cell interaction via anti-FN anti- 310 J Mammary Gland Biol Neoplasia (2010) 15:301–318 body or antibodies against β1 integrin subunit [124]. C expression is transient and largely restricted to embryo- Further, branching morphogenesis was rescued by adding genesis, yet it is upregulated in breast cancer [125, 133]. exogenous FN to siRNA-treated glands [124]. These Hence, TN-C has been described as an oncofetal protein. studies have clearly demonstrated that FN is required for TN-C was also demonstrated to be overexpressed in skin salivary gland branching morphogenesis, although, the during wound healing, particularly in the –epithelial question of whether this requirement is FN variant- junction [134]. In the mammary gland, TN-C is transiently specific is unknown [124]. expressed in dense stroma surrounding the budding Ovarian steroids are suggested to regulate FN expression epithelium during rodent embryogenesis, with little to no in the mammary gland [24]. Haslam and colleagues expression observed in the quiescent adult gland [135]. TN- measured a three-fold increase in FN protein levels around C expression is again upregulated during lactation and early mammary ducts between pre-puberty and sexual maturity postpartum involution [23, 136]. TN-C was found both in [24]. Moreover, ovariectomy of sexually-mature mice the milk of lactating mice and women [137, 138]. The resulted in a 70% decrease in the FN concentration in the function of TN during lactation is unclear, especially in mammary epithelium [24]. FN and the fibronectin receptor light of fact that β-casein expression was inhibited in α5β1 integrin are dramatically downregulated during late- mammary epithelial cells cultured on reconstituted base- pregnancy and lactation, and then upregulated during ment membrane containing exogenous TN-C [136]. mid-involution [23, 24]. Interestingly, the EDA isoform Another TN family member that was identified in the associated with pathological conditions in the liver is mammary gland is TN-X [62]. TN-X has only a developmentally regulated in the rat mammary gland tribrachion structure and is prominently expressed in testis similar to total FN, suggesting a role for EDA within the and muscle [139]. Burch and colleagues identified a normal mammary gland [23]. During the early stages of connective tissue defect in TN-X-deficient patients that involution, elevated matrix metalloproteinase (MMP) ac- involved hyperextensible skin and joints, vascular fragil- tivity correlated with higher levels of cleaved FN [23]. ity, and improper wound healing similar to Ehlers-Danlos Additionally, FN fragments can induce MMP9 activity in syndrome [140]. These observations suggest that TN-X mammary epithelial cells in vitro, suggesting a positive maintains tissue elasticity. During lactation the mammary feedback loop between FN fragments and MMP activity epithelial cells can experience considerable stretch forces, [23]. This has implications for clearance of the secretory and it is of interest to determine whether TN-X provides epithelium in involuting glands because FN fragments can such a function at this time. induce apoptosis in epithelial cells [58]. SPARC SPARC (secreted protein acidic and rich in cyste- The tenascin (TN) family consists of 5 members: ine, also known as ) has been identified in the TN-C, TN-R, TN-W, TN-X, and TN-Y. TN-C was the first mammary gland by gene array and mass spectrometry member of the TN family to be discovered, and is a six- based proteomics [52, 62]. SPARC is a small, 32 kDa armed glycoprotein with a 3D structure known as hexab- secreted glycoprotein that mediates cell-matrix interactions rachion. Each arm contains N-terminal structural domains [141]. SPARC contains a calcium-binding C-terminal for self-assembly, followed by EGF-like repeats, extracellular module, -like module, and N- alternative-spliced FN type III domains, and a C-terminal terminal acidic module [142, 143]. SPARC-null mice globular fibrinogen-homology domain [125]. The MW of exhibit abnormal collagen fibril morphology [144]. Using TNs ranges between 180 and 300 kDa, which may depend picrosirius red staining, the effect of SPARC on collagen on [125, 126]. TN is suggested to fibrillogenesis was evaluated in the dermis [144]. Com- display anti-adhesive properties via disruption of the pared to wild type mice, the dermis in SPARC-null mice interactions between the transmembrane heparan sulfate contained less striated collagen fibers [144]. Sage and receptor sydecan-4, α5β1 integrin receptor, and fibronectin colleagues found that SPARC preferentially binds to ECM [127, 128]. An anti-adhesive effect of TN-C on cells such proteins such as collagen I and LN111, but not to collagen as satellite cells has been shown to be context-dependent III [145]. They also demonstrated that SPARC displays an because other cells, for example, embryonic dorsal root epitope-specific anti-adhesive function that causes cell ganglion explants, were able to spread, migrate, and grow rounding [145]. In contrast, SPARC was shown to on TN-C ex vivo [129]. Disruption of the TN-C gene in positively regulate FN assembly and integrin-linked kinase mice did not produce any visible abnormalities [130]. activity, which resulted in FN-induced forma- Although, Kusakabe and colleagues demonstrated neuro- tion in lung fibroblasts [146]. SPARC has been reported to logical defects in TN-C null mice [131, 132]. Nonetheless, be developmentally upregulated within the mammary gland TN-C expression is associated with multiple tissues during during the transition between lactation and postpartum embryogenesis and pathological conditions. In general, TN- involution, correlating with increased levels of collagens J Mammary Gland Biol Neoplasia (2010) 15:301–318 311 and FN [23, 118]. Future research focused on SPARC discussed in previous sections [26]. Array data demon- function in collagen fibrillogenesis and FN assembly in a strate that decorin is upregulated during mammary gland dynamic organ such as the mammary gland is warranted. involution [52], where it likely contributes to the transient increase in collagen fibers [118]. Small Leucine-Rich Proteoglycan (SLRP) Biglycan is also a member of the SLRP class І SLRP family members are characterized by N-terminal family and is comprised of a core protein (~38 kDa) that cysteine-rich motifs and tandem leucine-rich repeats (LRR) is covalently attached to two GAG chains (chondroitin in their core protein. The core protein of SLRP is sulfate and/or ) with an overall MW of ‘decorated’ with one or more GAG chains. The SLRP gene 150–240 kDa [148]. Young and colleagues observed a family was categorized into five different classes based on reduction in bone formation and mass in biglycan-deficient several factors such as gene homology, distinctive N- mice [156]. Disruption of biglycan did terminal cyteine-rich motifs, and characteristic GAG side not cause upregulation of decorin expression and vice versa chains [147]. SLRPs were initially thought to only provide [151, 156, 157], although both biglycan and decorin belong structural support and participate in collagen fiber forma- to the same SLRP subfamily [157]. These results suggest tion. However, additional biological functions for SLRPs that decorin and biglycan have distinct, non-overlapping were recently identified [147]. SLRPs can directly bind to roles [156]. Schaefer et al. have tested the function of cell surface receptors and to growth factors, implicating biglycan in maintaining renal tissue elasticity under them in various signal transduction pathways and cellular pressure-induced injury caused by unilateral ureteral processes [147]. We will focus below on decorin and ligation [158]. They concluded that biglycan can induce biglycan from class І, as these SLRPs have been deter- -associated protein 1 expression in mined to be present in the mammary gland via gene array renal fibroblasts and mesangial cells, which subsequently and proteomics in multiple studies [52, 62]. help to retain elastic properties of the damaged tissue [158]. Their data is especially intriguing when it is Decorin Decorin core protein (~38 kDa) is covalently taken together with work from Salgado et al. who linked with a single chondroitin sulfate (CS) or dermatan demonstrated dynamic expression and localization of sulfate (DS) chain resulting in an overall MW of 90- decorin and biglycan in the mouse uterus during the estrous 140 kDa [148]. Decorin is a secreted protein and is cycle and pregnancy, suggesting that it is hormonally- expressed in various cell types such as fibroblasts and regulated [159, 160]. These studies raise the question of [149, 150]. Although decorin-deficient mice whether biglycan and decorin are also hormonally- experienced skin and lung fragility, possibly due to regulated in the mammary gland and whether they aberrant organization of fibrillar collagen, they are viable. influence the elastic properties of the gland during periods Variability in the shape and size of collagen fibrils in these of expansion. Moreover, biglycan transcript abundance in mice was observed compared to wild-type mice, as the mammary gland increased four- to five-fold during determined by EM of dermal collagen [151]. Based on the transition from lactation to involution [52, 118]. these studies, decorin has been suggested to be pivotal for Importantly, several members of the class II SLRP proper spatial alignment of stromal collagen fibers. family including , mimecan, and have Another function of decorin was demonstrated by Iozzo been identified in the mammary gland, as well as and colleagues, where decorin suppressed signaling evidence for their differential expression across the through ErbB2 in breast cancer cells in vitro and in reproductive cycle [52]. Future studies are required to xenograft models [152]. They suggested that decorin investigate the function of the class II SLRP family in the may inhibit EGFR signaling by initiating EGFR internal- mammary gland. ization and degradation by caveolar endocytosis [153]. Angiogenesis was also negatively affected by decorin as it Fibrous Connective Tissue downregulated vascular endothelial growth factor expres- sion in tumor cells [154]. When endothelial cells were The third structurally-distinct stroma in the mammary cultured in media from decorin-expressing tumor cells, gland is the relatively acellular fibrous connective tissue, they failed to migrate or form capillary-like structures in which is often found in large swaths (mm to cm range in vitro [154]. An additional role of decorin is to bind TGF- the rat and human) and is characterized by the absence β1 and sequester its activity in vitro [155]. Given that of epithelium and presence of fibroblasts, immune cells, decorin is crucial for proper fibrillar collagen organization, and high fibrillar collagen content (Fig. 2a, solid arrow one question concerns the role of decorin in mediating head). While the fibrous connective tissue is considered to tensional changes required for TGF-β1 activation as be separate from the intra- and interlobular stroma, these 312 J Mammary Gland Biol Neoplasia (2010) 15:301–318 compartments share many of the same ECM proteins such Future Directions as fibrillar collagens, FN, TN, SLRPs, and SPARC. However, a unique feature of fibrous connective tissue is Immune Cells and ECM Remodeling thepresenceofelasticfibers. The ECM of the mammary gland plays a role in the Elastic Fibers Elastic fibers provide structural support and infiltration and activation of immune cells. We have elasticity to various tissues. Elastic fibers are comprised of previously characterized involution as a wound healing a 31 kDa secreted protein called microfibril-associated microenvironment with high levels of alternatively activat- glycoprotein (MAGP) and another glycoprotein, the ed or M2 type macrophages [118]. How the immune cells 350 kDa fibrillin protein that assembles into 10- to 12-nm are recruited and ‘activated’ still remains unclear. However, [31]. Microfibrils then associate with other many of the proteins that make up the ECM can fragment elastic fiber constituents including , , and into matrikines and matricryptins, which have been dem- proteoglycans. The process of organizing elastic fibers in onstrated to influence leukocyte infiltration in other the connective tissue begins with the formation of the systems. Fragments of collagen I, collagen IV, LN and MAGP/fibrillin microfibrils that align parallel to fibroblasts nidogen-1 have all been shown to promote of and serve as tracks for the deposition of tropoelastin monocytes and neutrophils within the interstitial tissue, but (70 kDa), which is a soluble precursor of elastin. it remains unknown whether tissue-derived matrix frag- Tropoelastin is rich in hydrophobic amino acids and ments enter the blood stream to recruit immune cells or contains low amount of polar amino acids including several augment their motility upon arrival to the local tissue [118, lysine derivatives. The lysine derivatives are imperative for 168–171]. Once in the mammary gland, macrophages and covalent crosslinking between monomeric elastin via LOX neutrophils secrete including MMP9 and [31]. Microfibrils are described as a scaffold that facilitates that are able to breakdown the ECM [118, 172, 173]. the alignment of soluble monomeric elastin to coalesce and Without the influx of macrophages or neutrophils and their organize into amorphous elastin [31]. As elastic fibers crucial proteases, remodeling of the mammary tissue to its mature postnatally, elastin becomes the dominant compo- non-secretory postpartum state could potentially be delayed nent, with microfibrils being gradually displaced to the or incomplete. Werb and colleagues have evaluated the outer layer. The resultant fibers have physical properties importance of secreted proteases during involution in the that can withstand changes in the mechanical environment mammary gland by inhibiting a , plasma [161]. An additional constituent of elastic fibers is the kallikrein. They demonstrated that inhibition of this mast family (50–200 kDa), which have calcium-binding cell-associated protease during involution caused an accu- sites and consist of І, II, III domains with a central segment mulation of fibrillar collagen and delayed repopulation of that is composed of EGF-like modules. Fibulin 5 (66 kDa) adipocytes, thus preventing the gland from returning to its displays strong calcium-dependent binding to tropoelastin, pre-pregnant state [174]. Combined, these studies indicate while it weakly binds to carboxyl-terminal of fibrillin 1 that ECM turnover and immune cell function are highly [162]. Further, fibulin 5 knockout mice were generated to linked, creating a feedback loop that is necessary to analyze fibulin 5 function in elastic fiber formation in the complete gland remodeling after lactation. dermis. These mice demonstrated a requirement for fibulin In addition to facilitating immune cell infiltration, ECM 5 in tropoelastin deposition and potentially for optimal fragments may act as ligands to receptors present on LOX activity in vivo [162, 163]. Moreover, proteoglycans leukocytes in the mammary gland. Fragments of biglycan, such as biglycan and decorin were implicated in elasto- heparan sulfate, and hyaluronan have been shown to act as genesis by binding to tropoelastin, fibrillin 1, and MAGP ligands for toll like receptor 4 (TLR4). TLRs are part of the [158, 164, 165]. While much is known about the functions pattern recognition receptor family expressed on the cell of elastic fibers in other systems such as lung, skin, and surface of innate immune cells and dendritic cells. When a vascular system, their function in the mammary gland is ligand binds to a toll like receptor the immune cell becomes still elusive. Elastic fibers are anticipated to be particularly activated and/or secretes cytokines that can further activate important in the lactating mammary gland as the tensile cells of the adaptive immune system. Babelova et al. recently forces continuously change due to suckling, milk accumu- demonstrated that soluble biglycan binds to the TLR 2/4 on lation between nursing, and myoepithelial contraction on macrophages, stimulating them to synthesize and release the secretory alveolar epithelial cells. Further research on interleukin-1β, a proinflammatory cytokine [175]. Addition- the function and regulation of the fibrous connective tissue, ally, heparan sulfate and hyaluronan have been shown to including elastic fibers, is necessary given the correlations bind to the TLR4 on dendritic cells, resulting in dendritic cell between higher mammographic density, tissue tension, and maturation [176, 177]. 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